REMOVAL OF REPETITIVE SEQUENCES FROM FISH PROBES USING PCR-ASSISTED AFFINITY-CHROMATOGRAPHY

The vast majority of probes used in fluorescence in situ hybridization (FISH) contain repetitive DNA. This DNA is usually competed out of a hybridization reaction by the addition of an unlabeled blocking agent, Cot-1 DNA. We have successfully removed repetitive DNA from two compl ex FISH probe sets: a degenerate oligonucleotide-primed polymerase cha in reaction (DOP-PCR) single human chromosome library and genomic DNA. The procedure involved hybridizing in solution a DOP-PCR-amplifiable probe set with a 50-fold excess of biotin-labeled Cot-1 DNA, and captu ring the Cot-1 DNA-containing hybrids using streptavidin magnetic part icles, followed by purification and reamplification of the unbound fra ction. Probes were checked for depletion of repeats by hybridization t o chromosomes without Cot-1 DNA. Results showed hybridization patterns comparable to these achieved with untreated probes hybridized with Co t-1 DNA.